anti ref 2 p  (Bio-Rad)

Journal: bioRxiv

Article Title: Dredd-mediated cleavage of Kenny uncouples the IKK complex from selective autophagy to enable innate immunity

doi: 10.64898/2026.04.24.720600

Figure Lengend Snippet: (A) Adult Canton S flies were fed with 5 % sucrose and 100 µM chloroquine for 16 h. Endogenous Ref(2)P, Kenny and Atg8a levels in lysates from adult intestines were analysed with Western blotting with anti-Ref(2)P, anti-Kenny, anti-Atg8a and anti-Tubulin antibodies, n=3. (B) Adult Canton S flies were fed with 5% sucrose and Ecc15 for 16 h, Ref(2)P and Kenny levels were analysed in lysates from dissected intestines by Western blotting with anti-Ref(2)P, anti-Kenny, and anti-Actin antibodies. The relative protein levels of endogenous Ref(2)P and Kenny levels were quantified, n>4. (C) Adult Canton S and Atg8a-GFP flies were fed with 5% sucrose and Ecc15 for 16 h before whole flies were lysed and GFP-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-Kenny, anti-Atg8a, and anti-Actin antibodies. The ratio of immunoprecipitated Ref(2)P and Kenny to total Ref(2)P and Kenny was quantified, n=3. (D) Adult wildtype Canton S and GFP-Kenny expressing flies were fed with 5% sucrose and Ecc15 for 16 h before lysis. GFP-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-GFP, and anti-Actin antibodies. The ratio of immunoprecipitated Ref(2)P to total Ref(2)P was quantified, n=3.

Article Snippet: The following antibodies were used: anti-GFP (ab6556, Abcam), anti-HA (clone 3F10, #11867423001, Roche), anti-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad), anti-Ref(2)P (ab178440, Abcam), anti-Ref(2)P, and anti-Actin (C-11, sc-1615, Santa Cruz).

Techniques: Western Blot, Immunoprecipitation, Expressing, Lysis

Journal: bioRxiv

Article Title: Dredd-mediated cleavage of Kenny uncouples the IKK complex from selective autophagy to enable innate immunity

doi: 10.64898/2026.04.24.720600

Figure Lengend Snippet: (A) Dissected intestines from adult flies expressing mCherry-Atg8a (magenta) crossed with flies expressing HA-tagged wildtype or D21E mutant Kenny were stained for HA (yellow) and Ref(2)P (cyan). Co-localisation of Atg8a, Kenny and Ref(2)P is indicated as white puncta in the merged image. Midguts were imaged by confocal microscopy using at least 3 intestines per repeat, n=3. Scale bar 100 µm. (B) Adult control UbiGal4 flies and flies expressing HA-tagged wildtype or D21E mutant Kenny by control of UbiGal4 were fed with 5% sucrose and Ecc15 for 16 h before lysis. HA-immunoprecipitations were performed and the samples were analysed by Western blotting with anti-Ref(2)P, anti-HA and anti-Actin antibodies, n=5. The relative interaction between Ref(2)P and Kenny were quantified. (C) Adult control DaGal4 flies and flies expressing V5-tagged wildtype or Δ21 mutant Kenny by control of DaGal4 were lysed and Kenny expression levels were analysed with Western Blotting with anti-Kenny (upper panel), anti-V5 (middle panel), and anti-Actin antibodies, n>3.

Article Snippet: The following antibodies were used: anti-GFP (ab6556, Abcam), anti-HA (clone 3F10, #11867423001, Roche), anti-V5 (Clone SV5-Pk1, #MCA1360, Bio-Rad), anti-Ref(2)P (ab178440, Abcam), anti-Ref(2)P, and anti-Actin (C-11, sc-1615, Santa Cruz).

Techniques: Expressing, Mutagenesis, Staining, Confocal Microscopy, Control, Lysis, Western Blot

Journal: Cell Death & Disease

Article Title: Reduction of spermine synthase enhances autophagy to suppress Tau accumulation

doi: 10.1038/s41419-024-06720-8

Figure Lengend Snippet: A Lifespan of female flies with indicated genotype. n = 99, 116, 164, 110; Log-rank (Mantel–Cox) test. B Climbing performance of female flies with indicated genotype at indicated ages. n = 100, 100, 100, 100. C Staining of brains of 10 DAE flies with antibodies against Tau or cleaved caspase 3. The image is a representative of multiple brains in each group, n = 8, 9. D , E Quantification of the staining signal intensity of Tau ( D ) or cleaved caspase 3 ( E ) in ( C ). n = 8, 9. F Western blot of Tau, cleaved caspase 3, autophagy marker Atg8a and Ref(2)p in heads of 10 DAE flies. The image is a representative of multiple experiments. G – I Quantification of the protein level of Tau ( G ), cleaved caspase 3 ( H ) or autophagy markers ( I ) in ( F ). All the protein levels were normalized with the β-Actin level. All the values were further normalized by that of the control flies. J The ratio of Atg8a-II/Atg8a-I in ( F ). n = 6, 3, 6, 6 ( G – J ). B , H – J Two-way ANOVA Sidak’s multiple comparisons. D , E , G Student’s t test. Data represent mean ± SEM.

Article Snippet: The following commercially available antibodies were used: anti-GABARAP for Drosophila Atg8a probing (PM037, MBL) [ ], anti-Ref(2)P (ab178440, Abcam), anti-Tau (5A6, DSHB), anti-cleaved caspase 3 (9661, Cell Signaling), anti-LC3B (L7543, Sigma), anti-p62 (NBP1-48320, Novus Biologicals), anti-GFP (G5144, Invitrogen), anti-Actin (A1978, Sigma), Cy5-conjugated anti-HRP (123175021, Jackson ImmunoLab), and secondary antibodies conjugated to Alexa 488/568/647 (ThermoFisher Scientific) for brain immunohistochemical staining, or near-infrared (IR) dye 700/800 (Rockland) for western blot.

Techniques: Staining, Western Blot, Marker

Journal: Cell Death & Disease

Article Title: Reduction of spermine synthase enhances autophagy to suppress Tau accumulation

doi: 10.1038/s41419-024-06720-8

Figure Lengend Snippet: A Western blot of autophagy marker Atg8a and cargo recruiter Ref(2)p in 5 DAE flies. The image is a representative of multiple separate experiments. B Quantification of the protein level of Atg8a-I, Atg8a-II, and Ref(2)p in ( A ). All the protein levels were normalized with the β-Actin level. All the values were further normalized by that of the control flies. C The ratio of Atg8a-II/Atg8a-I in ( A ). n = 6; one-way ANOVA Tukey’s multiple comparisons. D Survival curve of 10 DAE female flies with indicated genotype under starvation. n = 50, 50, 25; Log-rank (Mantel–Cox) test. E Lifespan of female flies with indicated genotype. n = 105, 107, 91; Log-rank (Mantel–Cox) test. F Diagram of the polyamine metabolism pathway. G Polyamine levels of 10 DAE female flies with indicated genotype. Each dot indicates a sample of a homogenized mixture of ten flies. n = 3; one-way ANOVA Tukey’s multiple comparisons. Data represent mean ± SEM. The measurement showed here was done together with that showed in our previous publication . The data of the control and dSms −/− flies are shared in these two studies.

Article Snippet: The following commercially available antibodies were used: anti-GABARAP for Drosophila Atg8a probing (PM037, MBL) [ ], anti-Ref(2)P (ab178440, Abcam), anti-Tau (5A6, DSHB), anti-cleaved caspase 3 (9661, Cell Signaling), anti-LC3B (L7543, Sigma), anti-p62 (NBP1-48320, Novus Biologicals), anti-GFP (G5144, Invitrogen), anti-Actin (A1978, Sigma), Cy5-conjugated anti-HRP (123175021, Jackson ImmunoLab), and secondary antibodies conjugated to Alexa 488/568/647 (ThermoFisher Scientific) for brain immunohistochemical staining, or near-infrared (IR) dye 700/800 (Rockland) for western blot.

Techniques: Western Blot, Marker

Journal: bioRxiv

Article Title: The ubiquitin-conjugating enzyme UBE2D/eff maintains a youthful proteome and ensures protein quality control during aging

doi: 10.1101/2023.12.12.571303

Figure Lengend Snippet: (A) TMT mass spectrometry of Drosophila skeletal muscle from old versus young w 1118 flies at 8 vs. 1 week old; n=5 biological replicates. The x axis reports the Log2FC whereas the y axis reports the –log10( p -value). The E1 enzyme UBA1 and the E2 enzymes that are detected are indicated, including UBE2D/eff; n=5 (biological replicates). (B) The protein levels of UBE2D/eff significantly decline with aging (8 versus 1 week-old) in skeletal muscle; n=5 (biological replicates), SD, and p -values (Student’s t -test). ( C-D ) Immunostaining of Drosophila skeletal muscle at 3 weeks of age indicates that eff/UBE2D RNAi impairs protein quality control, as indicated by the higher age-related accumulation of aggregates of poly-ubiquitinated proteins, compared to control RNAi. The scale bar is 20 μm. In (C), n=10 (biological replicates), SD, and p -values (Student’s t -test). ( E-F ) Western blot analysis of detergent-soluble and insoluble fractions from Drosophila skeletal muscle indicates that eff RNAi impedes proteostasis, as indicated by higher levels of detergent-soluble and insoluble poly-ubiquitinated proteins compared to control RNAi (E). A similar increase is also found for the detergent- soluble levels of Ref(2)P/p62 (E). n=3 (biological replicates), SEM, ** p <0.01, ns=not significant (one-way ANOVA).

Article Snippet: The supernatants (Triton X-100 insoluble fraction) were collected and analyzed on 4-20% SDS-PAGE with anti-ubiquitin (Cell Signaling Technologies P4D1, #3936) and anti-Ref(2)P/p62 (Abcam #178840) antibodies.

Techniques: Mass Spectrometry, Immunostaining, Western Blot

Journal: bioRxiv

Article Title: The ubiquitin-conjugating enzyme UBE2D/eff maintains a youthful proteome and ensures protein quality control during aging

doi: 10.1101/2023.12.12.571303

Figure Lengend Snippet: ( A-B ) Western blot analysis of detergent-soluble and insoluble fractions from Drosophila skeletal muscle indicates that defects in proteostasis due to eff RNAi can be partially rescued by its human homolog UBE2D2, as indicated by the normalization of the detergent-soluble levels of poly-ubiquitinated proteins and Ref(2)P/p62. However, hUBE2D2 does not impact their detergent-insoluble levels, indicating that hUBE2D only partially rescues defects induced by eff/UBE2D RNAi. In (B), n=3 (biological replicates), SEM, ** p <0.01 (one-way ANOVA). ( C ) Muscle-specific eff/UBE2D RNAi reduces eff mRNA levels; n=3 (biological replicates), SEM, ** p <0.01 (one-way ANOVA). ( D ) Two distinct RNAi lines targeting eff/UBE2D reduce lifespan compared to control RNAi ( p <0.001, log-rank test). ( E ) The decline in organismal survival due to eff/UBE2D RNAi in muscle is partially rescued by transgenic hUBE2D expression compared to control mcherry overexpression ( p <0.001, log-rank test).

Article Snippet: The supernatants (Triton X-100 insoluble fraction) were collected and analyzed on 4-20% SDS-PAGE with anti-ubiquitin (Cell Signaling Technologies P4D1, #3936) and anti-Ref(2)P/p62 (Abcam #178840) antibodies.

Techniques: Western Blot, Transgenic Assay, Expressing, Over Expression

Journal: Communications Biology

Article Title: ACO2 deficiency increases vulnerability to Parkinson’s disease via dysregulating mitochondrial function and histone acetylation-mediated transcription of autophagy genes

doi: 10.1038/s42003-023-05570-y

Figure Lengend Snippet: a , b Expression levels of LC3 and p62 in the SN of mice were determined by Western blotting. c , d Quantification of LC3-II and p62 expression in the SN. n = 6. e Immunofluorescent staining of p62 (red) in the SN pc , TH + neurons were stained in green, and the nuclei were stained with DAPI (blue) (scale bars, 10 µm). n = 3. f , g Quantification of LC3 and Atg5 mRNA expression in the SN determined by qPCR. n = 3. h Expression levels of H3K9ac and H4K5ac in the SN of mice were determined by Western blotting. H3K9ac, H4K5ac and β-actin are derived from one membrane, whereas H3/H4 and GAPDH were derived from a separate membrane. i , j Quantification of H3K9ac and H4K5ac expression in the SN. n = 6. k , l Expression levels of GABARAP and p62/ref(2)P in fly heads determined by Western blotting. m , n Quantification of (K and L). n = 3–5. o Immunofluorescent staining of p62/ref(2)P (red) in DA neurons ( TH > GFP , green) of fly heads, the nuclei were stained with DAPI (blue) (scale bars, 10 µm, enlarged scale bars 2 µm). p Expression levels of H3K9ac and H4K5ac in fly heads were determined by Western blotting. q , r Quantification of H3K9ac and H4K5ac expression in fly heads. n = 3. s Climbing behavior of Rapa-treated flies. n = 6. Results are expressed as the mean ± SEM. *** P < 0.001, ** P < 0.01 and * P < 0.05. Statistical significance was performed with an unpaired t test between two groups, and a one-way ANOVA with Tukey’s post hoc test was used for comparing more than two groups. All the P values were two-sided. Source data are available as a Supplementary Data file.

Article Snippet: Antibodies: anti-TH antibody (1:1000, Millipore), anti-α-synuclein antibody (1:1000, CST), anti-LC3B antibody (1:1000, proteintech), anti-p62 antibody (1:2000, Abcam), anti-H3K9 antibody (1:5000, Active motif), anti-H4K5 antibody (1:5000, Millipore), anti-H3 antibody (1:1000, CST), anti-H4 antibody (1:1000, CST), anti-GABARAP antibody (1:1000, Abcam), anti-ref(2)P antibody (1:500, Abcam), anti-p-AMPK antibody (1:1000, Abcam), anti-p-ULK1 antibody (1:1000, CST), anti-PI3K III antibody (1:1000, CST), anti-p-mTOR antibody (1:1000, CST), anti-p-PI3K antibody (1:1000, CST), anti-ATG5 antibody (1:1000, Abcam) and anti-p-Beclin1 antibody (1:1000, CST), anti-DRP1 antibody (1:2000, proteintech), anti-MFN2 antibody (1:1000, Abcam), anti-β-actin antibody (1:5000, proteintech), anti-GAPDH antibody (1:5000, proteintech), peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Zhongshan Golden Bridge Biotechnology).

Techniques: Expressing, Western Blot, Staining, Derivative Assay, Membrane

Journal: Communications Biology

Article Title: ACO2 deficiency increases vulnerability to Parkinson’s disease via dysregulating mitochondrial function and histone acetylation-mediated transcription of autophagy genes

doi: 10.1038/s42003-023-05570-y

Figure Lengend Snippet: a ATP level in MES23.5 cells. The endogenous ATP level was detected by luminescent cell viability assay, and the data expressed as a percentage of the Con. n = 4. b The OCR measured in MES23.5 cells. c Expression levels of H3K9ac and H4K5ac in TA-treated MES23.5 cells were determined by Western blotting. Since H3K9ac and H4K5ac have the same molecular weight as H3 and H4, respectively, we performed experiments on two SDS-PAGE gels separately and provide the results of two membranes. d Expression level of LC3 in TA-treated MES23.5 cells were determined by Western blotting. e , f Quantification of ( c ), n = 5. g Quantification of ( d ), n = 4. h , i Quantification of LC3 and Atg5 mRNA expression in MES23.5 cells determined by qPCR. n = 3. j Climbing behavior of CoQ10-treated flies. n = 6. k , l Expression levels of GABARAP and p62/ref(2)P in fly heads determined by Western blotting. m , n Quantification of ( k and l ). n = 3. o , p Quantification of LC3 and Atg5 mRNA expression in fly heads determined by qPCR. n = 3. Results are expressed as the mean ± SEM. **** P < 0.0001, *** P < 0.001, ** P < 0.01 and * P < 0.05. Differences among means were determined by one-way ANOVA followed by the Tukey’s test for post-hoc comparisons. All the P values were two-sided. Source data are available as a Supplementary Data file.

Article Snippet: Antibodies: anti-TH antibody (1:1000, Millipore), anti-α-synuclein antibody (1:1000, CST), anti-LC3B antibody (1:1000, proteintech), anti-p62 antibody (1:2000, Abcam), anti-H3K9 antibody (1:5000, Active motif), anti-H4K5 antibody (1:5000, Millipore), anti-H3 antibody (1:1000, CST), anti-H4 antibody (1:1000, CST), anti-GABARAP antibody (1:1000, Abcam), anti-ref(2)P antibody (1:500, Abcam), anti-p-AMPK antibody (1:1000, Abcam), anti-p-ULK1 antibody (1:1000, CST), anti-PI3K III antibody (1:1000, CST), anti-p-mTOR antibody (1:1000, CST), anti-p-PI3K antibody (1:1000, CST), anti-ATG5 antibody (1:1000, Abcam) and anti-p-Beclin1 antibody (1:1000, CST), anti-DRP1 antibody (1:2000, proteintech), anti-MFN2 antibody (1:1000, Abcam), anti-β-actin antibody (1:5000, proteintech), anti-GAPDH antibody (1:5000, proteintech), peroxidase-conjugated anti-mouse or anti-rabbit IgG (1:5000, Zhongshan Golden Bridge Biotechnology).

Techniques: Cell Viability Assay, Expressing, Western Blot, Molecular Weight, SDS Page